Cell differentiation depends fundamentally on the turnover of selected regulatory and structural proteins. This universal aspect of development is mediated in large part by polyubiquitination, which targets proteins for disposal in the 26S-proteasome. A major group of polyubiquitin ligases, the SCF (Skp1-cullin1-Fbox) subclass of cullin-RING-type E3 ubiquitin (Ub)-ligases (CRLs), has been directly implicated in numerous physiological (including cell cycle) and developmental processes. A critical feature is their use o a specificity factor for selecting targets, following a priming event such as phosphorylation. Considerable evidence indicates that CRL Ub-ligases are also regulated. Our findings in the model organism Dictyostelium reveal new and novel mechanisms for regulating the SCF class itself. These mechanisms appear to be widespread in protists including many important human pathogens. The novel mechanisms involve covalent modification of the Skp1 adaptor by prolylhydroxylation and subsequent serial modification by 5 sugars to ultimately form a pentasaccharide. We defined the genetics and enzymology of the pathway in the last and earlier project periods. These studies also revealed striking changes in the O2 dependence of development which we traced back to an O2 sensor function of the prolyl 4-hydroxylase analogous to a corresponding process in humans. We also discovered that successive glycosylation steps modulate O2 sensing, and that the final glycosyltransferase in the pathway, AgtA, has enzyme-independent functions that are also necessary for proper development. Our newest findings now suggest that these modifications alter the conformation of Skp1, which inhibits its homodimerization and promotes binding of Skp1 to Fbox proteins, leading to their auto-polyubiquitination and premature degradation. Furthermore, AgtA competitively binds unmodified Skp1, by a novel self-limiting mechanism mediated by glycosylation. Because these interactions pertain to the assembly of E3SCFUb-ligases, we hypothesize that the Skp1 modification enzymes ultimately control the ubiquitination of many of the ~50 predicted Fbox proteins, most of which are differentially regulated during development, and potentially their target substrates as well. The goal of this project is to define the biochemical mechanism of how this occurs. This model is important be- cause it offers a novel mode of specific regulation of E3SCFUb-ligases with major impact on development, and the occurrence of the 6-enzyme pathway in pathogenic protists presents a large drug target for future exploitation. The studies will be conducted in Dictyostelium, an experimentally facile organism where we have developed invaluable tools to test the hypothesis. Aim 1 will investigate how hydroxylation, glycosylation, and AgtA affect assembly of SCF complexes and their E3 Ub-ligase activities in vitro. Aim 2 will investigate the relevance of the findings to Skp1 complex assembly and activities in cells using immunoprecipitation and microscopy. Aim 3 will employ gene and inhibitor synthetic studies to address the linkage of Skp1 modification to ubiquitination and degradation activities in developmental regulation.